藤黄酸激活泛素连接酶CHIP逆转ABCB1介导的上皮源性肿瘤多药耐药性研究

Multidrug resistance (MDR) caused by the ABCB1 protein during the cancer treatment process is an important issue faced by the oncologists. Our previous studies have shown that Gambogic acid (GA) reduce the ABCB1 protein levels through the proteasome pathway in the MDR cells. However, the mechanism of the ABCB1 protein degradation remained unclear. The project intends to further research whether GA can promote the interaction between ubiquitin ligase CHIP and ABCB1 in tumor cells, leading to ABCB1 ubiquitination and degradation. In this study we will examine the ABCB1 activity and cellular chemotherapeutic drug concentrations in MDR cells by fluorescence intensity detection after GA incubation; detect GA-activated interaction between CHIP and ABCB1 ubiquitination by co-immunoprecipitation method; observe the drug combination to inhibit tumor growth in nude mice to evaluate chemotherapy sensitivity; verify the interaction between CHIP and ABCB1 in clinical tumor samples. The significance of this project is to probe CHIP reversing MDR mechanisms; develop GA as a lead compound of novel ABCB1 inhibitor; explore new pharmacological activity of GA for drug combination reference in the clinical trials of GA.

肿瘤治疗过程中由ABCB1蛋白引起的多药耐药性是肿瘤医生面临的重要问题。我们前期研究显示藤黄酸能通过蛋白酶体途径降低细胞内ABCB1蛋白水平，提高化疗药物在细胞内浓度，但是其中的ABCB1蛋白降解机制尚不清楚。本项目拟进一步研究藤黄酸是否可促进肿瘤细胞中泛素连接酶CHIP和ABCB1相互作用，从而引起ABCB1泛素化而降解。本研究将采用荧光强度测试方法检测藤黄酸孵育的耐药细胞株中ABCB1活性及细胞内化疗药物浓度；免疫共沉淀方法检测藤黄酸激活的CHIP蛋白对ABCB1的泛素化作用；在荷瘤裸鼠上观察药物联合应用抑制肿瘤生长的效果，评价藤黄酸的化疗增敏作用；在临床肿瘤样本中验证CHIP与ABCB1的相互作用。本项目的研究意义在于以藤黄酸为探针研究CHIP逆转肿瘤多药耐药性的机制；藤黄酸可为新型ABCB1抑制剂开发提供先导结构；藤黄酸的新药理活性还将为藤黄酸的临床试验研究提供药物联合使用参考。

虽然有研究报道了藤黄酸与其它一些化疗药物的协同作用，但是尚未见到有藤黄酸逆转由ABCB1引起的肿瘤多药耐药性。本研究发现藤黄酸在低于其诱导细胞凋亡剂量上对肿瘤多药耐药性的逆转作用及相关机制。采用体外测试ABCB1活性、检测ABCB1高表达细胞耐药性和ABCB1荧光底物排出能力、构建荷瘤裸小鼠观察药物合用抑制肿瘤生长效果等手段研究藤黄酸抑制ABCB1活性而发挥化疗增敏作用。我们发现藤黄酸显著抑制重组表达的ABCB1蛋白活性，能够增加多药耐药细胞中ABCB1底物的浓度。而且藤黄酸通过CHIP介导的泛素蛋白酶体途径降解了ABCB1蛋白。我们也发现藤黄酸能在离体和整体动物模型中协同增强化疗药物对肿瘤细胞的生长抑制作用。本研究的意义在于发现藤黄酸的新药理作用，为藤黄酸的临床药物组合方案应用提供依据；而且本研究的发现将有利于开发以藤黄酸作为先导结构的新型ABCB1抑制剂。